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Targeting Super-Enhancer-Associated Oncogenes in Oesophageal

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Targeting Super-Enhancer-Associated Oncogenes in Oesophageal Downloaded from http://gut.bmj.com/ on May 23, 2016 - Published by group.bmj.com Gut Online First, published on May 10, 2016 as 10.1136/gutjnl-2016-311818 Oesophagus ORIGINAL ARTICLE Targeting super-enhancer-associated oncogenes in oesophageal squamous cell carcinoma Yan-Yi Jiang,1 De-Chen Lin,1,2 Anand Mayakonda,1 Masaharu Hazawa,1 Ling-Wen Ding,1 Wen-Wen Chien,1 Liang Xu,1 Ye Chen,1 Jin-Fen Xiao,1 William Senapedis,3 Erkan Baloglu,3 Deepika Kanojia,1 Li Shang,4 Xin Xu,4 Henry Yang,1 Jeffrey W Tyner,5 Ming-Rong Wang,4 H Phillip Koeffler1,6,7 ▸ Additional material is ABSTRACT published online only. To view Objectives Oesophageal squamous cell carcinoma Significance of this study please visit the journal online (http://dx.doi.org/10.1136/ (OSCC) is an aggressive malignancy and the major gutjnl-2016-311818). histological subtype of oesophageal cancer. Although recent large-scale genomic analysis has improved the What is already known on this subject? description of the genetic abnormalities of OSCC, few ▸ fi The genomic landscape of oesophageal For numbered af liations see targetable genomic lesions have been identified, and no end of article. squamous cell carcinoma (OSCC) has been molecular therapy is available. This study aims to identify established; however, genetic alterations of Correspondence to druggable candidates in this tumour. actionable targets are infrequent in this Dr Yan-Yi Jiang, Cancer Design High-throughput small-molecule inhibitor malignancy. Science Institute of Singapore, screening was performed to identify potent anti-OSCC ▸ Super-enhancers (SEs) recruit an exceptionally National University of Singapore, NUS, CSI, MD6 14 compounds. Whole-transcriptome sequencing (RNA-Seq) large number of transcription factors/cofactors, Medical drive #13-01, and chromatin immunoprecipitation sequencing (ChIP- and they differ from typical enhancers in size, Singapore 119260, Singapore; Seq) were conducted to decipher the mechanisms of transcription factor density and ability to [email protected] action of CDK7 inhibition in OSCC. A variety of in vitro induce transcription. Dr De-chen Lin, Sun Yat-Sen and in vivo cellular assays were performed to determine ▸ SEs are found to be associated with key Memorial Hospital, Sun Yat- the effects of candidate genes on OSCC malignant lineage-specific master regulators in normal Sen University, Guangzhou phenotypes. somatic cells as well as with a few critical 510120, China; Results The unbiased high-throughput small-molecule [email protected] oncogenes in several types of tumour cells. inhibitor screening led us to discover a highly potent anti- Dr Ming-Rong Wang, Cancer OSCC compound, THZ1, a specific CDK7 inhibitor. RNA- What are the new findings? Institute/Hospital, Peking Union Seq revealed that low-dose THZ1 treatment caused ▸ The SE landscape is established in OSCC cells, Medical College and Chinese and many SE-associated, squamous-specific Academy of Medical Sciences, selective inhibition of a number of oncogenic transcripts. Beijing 100021, China; Notably, further characterisation of the genomic features master regulators and novel oncogenic [email protected] of these THZ1-sensitive transcripts demonstrated that they transcripts are identified. were frequently associated with super-enhancer (SE). ▸ Targeting SE-associated transcription activation Y-YJ, D-CL and AM have by a small-molecule CDK7 inhibitor, THZ1, contributed equally. Moreover, SE analysis alone uncovered many OSCC lineage-specific master regulators. Finally, integrative shows powerful antineoplastic properties Received 8 March 2016 analysis of both THZ1-sensitive and SE-associated against OSCC cells. Revised 11 April 2016 transcripts identified a number of novel OSCC oncogenes, ▸ PAK4 is an SE-associated candidate drug target Accepted 20 April 2016 including PAK4, RUNX1, DNAJB1, SREBF2 and YAP1, in OSCC. with PAK4 being a potential druggable kinase. How might it impact on clinical practice in Conclusions Our integrative approaches led to a the foreseeable future? catalogue of SE-associated master regulators and ▸ fi Through characterising the transcriptional oncogenic transcripts, which may signi cantly promote abnormalities in OSCC, our study suggests that both the understanding of OSCC biology and the targeting transcriptional activation programme development of more innovative therapies. rather than specific genomic lesions might provide better efficacy in the clinical management of solid tumours with highly INTRODUCTION complex genomes, such as OSCC. Oesophageal squamous cell carcinoma (OSCC) is ▸ This work may help establish the potential one of the most common and aggressive GI malig- therapeutic merit of targeting SE-associated nancies.12Due to a lack of understanding of the oncogenic transcription programme through a molecular basis and limited treatment options, the small-molecule CDK7 inhibitor, THZ1, for the prognosis for patients with OSCC has not treatment of patients with OSCC. To cite: Jiang Y-Y, Lin D-C, improved for decades.3 Recently, researchers, Mayakonda A, et al. Gut Published Online First: including ourselves, have determined the genomic 4–8 [please include Day Month landscape of OSCC and identified a number of except those affecting PIK3CA and FGFR1. Year] doi:10.1136/gutjnl- driver events; however, genetic alterations of drug Clearly, alternative molecular approaches are 2016-311818 targets are infrequent in patients with OSCC, needed to further elucidate the pathogenesis of Jiang Y-Y, et al. Gut 2016;0:1–11. doi:10.1136/gutjnl-2016-311818 1 Copyright Article author (or their employer) 2016. Produced by BMJ Publishing Group Ltd (& BSG) under licence. Downloaded from http://gut.bmj.com/ on May 23, 2016 - Published by group.bmj.com Oesophagus OSCC for developing more innovative and effective regimens. metastasised into the lungs (around 45 days after initial injec- Transcription factors and cofactors interact with enhancers to tion), they were randomly separated into two groups and control specific gene expression programmes, which are funda- treated with either vehicle or THZ1 (twice daily, 10 mg/kg). At mental to cell biology. Recently, a special group of enhancers, the end of metastasis assays, mice were euthanised and exam- termed super-enhancers (SEs),910have been identified in many ined. Metastasis nodules in lung tissues were fixed in Bouin’’s cell types. SEs recruit an exceptionally large number of tran- solution, embedded in paraffin, cut into 5 mm sections and scription factors/cofactors, and they differ from typical enhan- stained with H&E. All of the animal experiments in this study cers (TEs) in size, transcription factor density and ability to were approved by the Institutional Animal Care and Use induce transcription.11 12 SEs are frequently associated with key Committee (IACUC), National University of Singapore. lineage-specific genes that control cell state and differentiation in normal somatic cells.13 Interestingly, SEs are also found to High-throughput small-molecule inhibitor screening drive the expression of a few critical oncogenes in several types Cell lines were screened for sensitivity against a panel of 104 of tumour cells, such as STAT3,10 MYCN,14 and TAL1.15 small-molecule inhibitors as previously described.17 Briefly, cells However, little is known regarding whether and how SEs drive were exposed to graded concentrations of each drug in 384 well the pathogenesis of OSCC. plates with a seeding density of 250 cells per well in 50 μL final Interestingly, expression of a few SE-associated oncogenes was volume per well. Each drug was tested across an eight-point, reported to be particularly vulnerable to transcriptional inhib- threefold interval dose–response curve (including the no-drug ition, offering a potential cancer targeting approach.12 16 One of control). After 3 days, relative abundance of viable cells was the possible mechanisms is that these SE-associated oncogenes quantified in each well using a tetrazolium-based MTS assay. All are addicted to continuous active transcription, allowing for absorbance values from the MTS assay were normalised to the selective effects before a global blockade of transcription is average of 48 wells per plate containing no drug, and these nor- achieved. malised values were used to fit a third-order polynomial curve In this study, we performed high-throughput small-molecule to each drug dose–response. IC50 values were interpreted from inhibitor screening, and identified THZ1, a newly developed this curve fit to assess the relative sensitivity of each cell line to covalent CDK7 inhibitor,16 as a potent anti-OSCC compound. each drug. Interestingly, we observed that low-dose THZ1 treatment eli- cited selective effects against genes significantly enriched in pro- Chromatin immunoprecipitation sequencing data analysis cesses important in cancer biology. We further characterised the Chromatin immunoprecipitation sequencing (ChIP-Seq) reads SE landscape in OSCC cells and found that THZ1-sensitive were aligned to human reference genome (build GRCh37/hg19) transcripts were significantly more frequently associated with using Bowtie Aligner. ChIP-Seq peaks were identified using SEs. Finally, through integrative analysis of the gene transcrip- MACS (Model-Based Analysis of ChIP-seq) by considering reads tion signature and SE features, we established a functional mapped only once at a given locus. Wiggle files were generated genomic approach to discover novel oncogenes in OSCC. using read pileups for every 50 base pair bins. These wiggle files were normalised
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